Phage Therapy for Drug-Resistant Pathogens

Bacteriophages

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In this project, I will be researching phage therapy as a defense against drug-resistant pathogens, specifically bacteria. Typically, antibiotics are used in order to combat bacterial infections and have had a lot of success. Although, when antibiotics are used often, bacteria is capable of developing a resistance to the drug, rendering it much less effective. A newer potential defense of pathogens is phage therapy. Phage therapy uses bacteriophages, which are viruses that target and infect bacteria. Each type of bacteria is susceptible to a number of bacteriophages . When using a bacteriophage to target a pathogen, bacterial lysis often occurs, leading to the breakdown of a cell’s membrane and bursting of the cell . This would be a successful result in targeting a pathogen. The use of phage therapy actually dates back to the early 1900s, even before antibiotics were discovered. Once antibiotics were widely used, phage therapy dissipated but now that bacteria are developing drug-resistant characteristics, the use of phage therapy has gained a renewed interest and is continuously expanding.

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Bacteriophage Function

Bacteriophages are known as viruses that specifically infect bacteria. In terms of human health, bacteriophages are present in the intestinal community, which help with digestion, immune system function, and mental health .

In order for bacteriophages to function in combating bacteria, they must begin an infection cycle. To start, they attach to the surface of a host cell, which is allowed by certain proteins on the host cell surface, called cell-surface receptors . This protein described actually performs a very important function for the host cell, but the bacteriophage has evolved to use it to its advantage, such as attachment. Once attached, the phage injects its genome into the cell through the cell envelope. Once the phage’s DNA is inside the cell, replication in a lytic cycle begins. As many progeny phages are assembled as possible in this lytic cycle . Not only are the phage genomes replicated, but the corresponding enzymes and proteins are also assembled. Once enough progeny phages are created, the host cell lyses, meaning that the cell wall bursts which releases the progeny . The phage then inserts its DNA into the host cell’s cytoplasm, leading to the expression of phage genes by the host cell RNA polymerase and ribosomes . Phage genomes are continuously replicated, along with enzymes and ribosomes that then produce the phage capsid proteins. Lastly, the phage genome expresses an enzyme that lyses the host cell wall, which releases these completed virus particles . With the destruction of the host cell wall, this leads to the destruction of the cell, and therefore the bacteria, making it clear that bacteriophages can be instrumental in the killing of bacteria.

Antibiotic Resistance and Phage Therapy Application

Antibiotics have been widely used to combat bacterial infections, although it is well known that bacteria are capable of developing resistance to certain antibiotic medications. This is a major threat to global health due to the increasing antibiotic resistance levels due to antibiotic overuse. There are various mechanisms that lead to antibiotic resistance in pathogens, such as drug target modification, molecular bypass, active efflux, and the chemical modification of the compound . In drug target modification, it is possible that a point mutation occurs in specific genes, which can change the amino acid that is present, leading to an alteration in protein structure, preventing the binding of antibiotics and therefore antibiotic function . In molecular bypass, microbes can avoid antibiotic action through specific avoidance mechanisms. For example, in vancomycin resistance, the substitution of an amide bond of D-Ala-D-Ala with an ester linkage gets rid of a hydrogen bond donor, causing electronic repulsion which therefore prevents binding of the antibiotic, one example of avoiding antibiotic function without mutations . There is also active efflux, which is essentially the removal of antibiotics from the cell. Proteins are in charge of this removal, along with proton pumps . Lastly, in chemical modification, enzymes catalyze the inactivation of specific antibiotics . Through the various, complex mechanisms of antibiotic resistance, it is clear that other manners of pathogen defense will be necessary to combat these antibiotic-resistance bacteria.

One possibility when exploring alternatives to antibiotics in bacterial defense is phage (bacteriophage) therapy. Phage therapy was actually first described in 1907 by Félix d’Herelle in France, even before antibiotics were discovered . It was explained that every type of bacteria is susceptible to a certain limited number of specific types of phages . Due to the fact that when bacteria are infected by bacteriophages, it often leads to lysis, this technique was considered feasible for the treatment of bacterial infections, as long as the phage specific was to the pathogen . There was some success and some failure when the experimentation began, as phages were used to treat various illnesses such as cholera or typhoid fever, by giving a patient phage in an open wound, or orally provided through aerosol or injection . As soon as antibiotics were discovered and had success, phage therapy was placed aside, but it has become much more relevant today with the increase in antibiotic resistance.

Phage Therapy Approaches and Applications

There are various approaches to phage therapy delivery, one being monophage therapy, which points to the use of a single phage. Monophage therapy has seen success in the laboratory setting, specifically with animals . Although it has been successful, this technique is hampered by a few drawbacks, one being that phages must be specific to the bacteria, so pinpointing one singular phage may cause difficulty . Another disadvantage is the possibility of bacteria becoming resistant to the phage in monophage therapy. This highlights the importance of polyphage therapy, where multiple different phages are used in a phage cocktail . The evolution of bacterial resistance can happen relatively quickly, and therefore it is important that this is avoided as much as possible [5]. A phage cocktail is often used, which combines phages in order to target multiple strains of the same bacterial species, or multiple species [5]. Although essential to avoid bacterial resistance, using multiple phages may lead to complexities in production or safety questions. Due to the fact that phages are specific to each bacteria, it is also important to not include a random mixture of phages, but instead an optimized phage usage that is specific to the knowledge of bacterial receptors [5]. One example of this would be using phages that each target a different receptor in the same bacterial host, which in turn will reduce the likelihood and speed of phage resistance. Another strategy to minimize resistance is by combining both monophage therapy and polyphage therapy in a sequential order <ref name=dd/. By administering monophage and polyphage therapies at different times in treatment, the bacteria will be continuously exposed to new phages, making it harder to develop resistance, while also keeping the bacteria at a low level so that the immune system can take effect. Currently, phage cocktails are available as over-the-counter medicine in Russia and Georgia for bacterial infections <ref name=dd/. Two well-known generic phage cocktails, Pyophage (PYO) and Intestiphage have been used. Pyophage targets at least 6 common bacteria, while Intestiphage targets about 23 different enteric, or disease-causing, bacteria <ref name=dd/. Although not yet approved in Western countries, the study of polyphage therapy effectiveness has increased and continues to be subjective to clinical studies.

It is often very effective in a clinical setting to use more than one medicinal therapy in fighting a pathogen. It has commonly been in practice to combine phage therapy with antibiotic use in order to combat bacterial infections. The term phage-antibiotic synergy (PAS) has been used to describe the combinations of phage therapy and antibiotic use <ref name=dd/. This has been used in previous studies, showing that while using antibiotics, this could increase the bacterial production of lytic phages <ref name=dd/. There is even more potential for this strategy for a variety of bacterial species in the order of treatment. It has been demonstrated that treatment with bacteriophages prior to antibiotics may lead to the most efficient killing of bacteria <ref name=dd/. Optimizing the timing of phage-antibiotic synergy may be successful in improving efficacy <ref name=dd/. By using a combination of phages and antibiotics, this limits the amount of antibiotic used and therefore decreases the likelihood of developed resistance <ref name=dd/. Secondly, a combination of the two treatments would give antibiotics a restored strength against bacteria <ref name=dd/. The effectiveness of the combination treatment has been demonstrated in broiler chickens infected with E. coli [6]. With solely an antibiotic, specifically enrofloxacin, mortality was decreased by 65%. Phage treatment alone decreased mortality by 53% [6]. The combination of phage therapy and enrofloxacin resulted in no mortality, demonstrating the clear effectiveness of phage-antibiotic synergy treatment [6]. Another study observed a 10,000-fold greater reduction in bacterial load with phage and ciprofloxacin rather than each treatment alone in rats with P. aeruginosa [6]. There have been very few clinical trials experimenting with phage-antibiotic synergy, but the combination has demonstrated significant potential in the treatment of bacterial infections in vivo.

Genetic modification has demonstrated large potential in terms of the medical field, and specifically with bacteriophage treatment. In genetically modifying pre-existing biological systems to perform tasks that are not typically done are one way that phage therapy may progress in the future. In bioengineering phages, they can become more dynamic in circumstances such as expanded host range, switching host tropism, or modification of phage capsids <ref name=dd/. One study was able to genetically modify the host range of E. coli phage T2 by incorporating long tail fiber genes of a different phage through homologous recombination <ref name=dd/. In doing so, the host range of E. coli phage T2 expanded, while also keeping its success in lytic activity <ref name=dd/, demonstrating successful usage of bioengineering in phage therapy. Another study altered E. coli phage T7 to express the enzyme dispersin B, which can degrade a key component of bacterial biofilms <ref name=dd/. This engineered virus was able to reduce biofilm cell counts by >100-fold compared to the original, wild-type phage

Authored for BIOL 238 Microbiology, taught by Joan Slonczewski, 2021, Kenyon College.